This invention relates to RNA polymerase III-based methods and systems for expression of therapeutic RNAs in cells in vivo or in vitro.
The RNA polymerase III (pol III) promoter is one found in DNA encoding 5S, U6, adenovirus VA1, Vault, telomerase RNA, tRNA genes, etc., and is transcribed by RNA polymerase III (for a review see Geiduschek and Tocchini-Valentini, 1988 Annu. Rev. Biochem. 57, 873-914; Willis, 1993 Eur. J. Biochem. 212, 1-11). There are three major types of pol III promoters: types 1, 2 and 3 (Geiduschek and Tocchini-Valentini, 1988 supra; Willis, 1993 supra) (see FIG. 1). Type 1 pol III promoter consists of three cis-acting sequence elements downstream of the transcriptional start site a) 5′ sequence element (A block); b) an intermediate sequence element (I block); c) 3′ sequence element (C block). 5S ribosomal RNA genes are transcribed using the type 1 pol III promoter (Specht et al., 1991 Nucleic Acids Res. 19, 2189-2191.
The type 2 pol III promoter is characterized by the presence of two cis-acting sequence elements downstream of the transcription start site. All Transfer RNA (tRNA), adenovirus VA RNA and Vault RNA (Kikhoefer et al., 1993, J. Biol. Chem. 268, 7868-7873) genes are transcribed using this promoter (Geiduschek and Tocchini-Valentini, 1988 supra; Willis, 1993 supra). The sequence composition and orientation of the two cis-acting sequence elements-A box (5′ sequence element) and B box (3′ sequence element) are essential for optimal transcription by RNA polymerase III.
The type 3 pol III promoter contains all of the cis-acting promoter elements upstream of the transcription start site. Upstream sequence elements include a traditional TATA box (Mattaj et al., 1988 Cell 55, 435-442), proximal sequence element (PSE) and a distal sequence element (DSE; Gupta and Reddy, 1991 Nucleic Acids Res. 19, 2073-2075). Examples of genes under the control of the type 3 pol III promoter are U6 small nuclear RNA (U6 snRNA) and Telomerase RNA genes.
In addition to the three predominant types of pol III promoters described above, several other pol III promoter elements have been reported (Willis, 1993 supra) (see FIG. 1). Epstein-Barr-virus-encoded RNAs (EBER), Xenopus seleno-cysteine tRNA and human 7SL RNA are examples of genes that are under the control of pol III promoters distinct from the aforementioned types of promoters. EBER genes contain a functional A and B box (similar to type 2 pol III promoter). In addition they also require an EBER-specific TATA box and binding sites for ATF transcription factors (Howe and Shu, 1989 Cell 57,825-834). The seleno-cysteine tRNA gene contains a TATA box, PSE and DSE (similar to type 3 pol III promoter). Unlike most tRNA genes, the seleno-cysteine tRNA gene lacks a functional A box sequence element. It does require a functional B box (Lee et al., 1989 J. Biol. Chem. 264, 9696-9702). The human 7SL RNA gene contains an unique sequence element downstream of the transcriptional start site. Additionally, upstream of the transcriptional start site, the 7SL gene contains binding sites for ATF class of transcription factors and a DSE (Bredow et al., 1989 Gene 86, 217-225).
Gilboa WO 89/11539 and Gilboa and Sullenger WO 90/13641 describe transformation of eucaryotic cells with DNA under the control of a pol III promoter. They state:                “In an attempt to improve antisense RNA synthesis using stable gene transfer protocols, the use of pol III promoters to drive the expression of antisense RNA can be considered. The underlying rationale for the use of pol III promoters is that they can generate substantially higher levels of RNA transcripts in cells as compared to pol II promoters. For example, it is estimated that in a eucaryotic cell there are about 6×107 t-RNA molecules and 7×105 mRNA molecules, i.e., about 100 fold more pol III transcripts of this class than total pol II transcripts. Since there are about 100 active t-RNA genes per cell, each t-RNA gene will generate on the average RNA transcripts equal in number to total pol II transcripts. Since an abundant pol II gene transcript represents about 1% of total mRNA while an average pol II transcript represents about 0.01% of total mRNA, a t-RNA (pol III) based transcriptional unit may be able to generate 100 fold to 10,000 fold more RNA than a pot II based transcriptional unit. Several reports have described the use of pol III promoters to express RNA in eucaryotic cells. Lewis and Manley and Sisodia have fused the Adenovirus VA-1 promoter to various DNA sequences (the herpes TK gene, globin and tubulin) and used transfection protocols to transfer the resulting DNA constructs Into cultured cells which resulted in transient synthesis of RNA in the transduced cell. De la Pena and Zasloff have expressed a t-RNA-Herpes TK fusion DNA construct upon microinjection into frog oocytes. Jennings and Molloy have constructed an antisense RNA template by fusing the VA-1 gene promoter to a DNA fragment derived from SV40 based vector which also resulted in transient expression of antisense RNA and limited inhibition of the target gene”. [Citations omitted.]        
The authors describe a fusion product of a chimeric tRNA and an RNA product (see FIG. 1C of WO 90/13641). In particular they describe a human tRNA meti derivative 3-5. 3-5 was derived from a cloned human tRNA gene by deleting 19 nucleotides from the 3′ end of the gene. The authors indicate that the truncated gene can be transcribed if a termination signal is provided, but that no processing of the 3′ end of the RNA transcript takes place.
Adeniyi-Jones et al., 1984 Nucleic Acids Res. 12, 1101-1115, describe certain constructions which “may serve as the basis for utilizing the tRNA gene as a ‘portable promoter’ in engineered genetic constructions.” The authors describe the production of a so-called Δ3′-5 in which 11 nucleotides of the 3′-end of the mature tRNAimet sequence are replaced by a plasmid sequence, and are not processed to generate a mature tRNA. The authors state:                the properties of the tRNAimet 3′ deletion plasmids described in this study suggest their potential use in certain engineered genetic constructions. The tRNA gene could be used to promote transcription of theoretically any DNA sequence fused to the 3′ border of the gene, generating a fusion gene which would utilize the efficient polymerase III promoter of the human tRNAimet gene. By fusion of the DNA sequence to a tRNAimet deletion mutant such as Δ3′-4, a long read-through transcript would be generated in vivo (dependent, of course, on the absence of effective RNA polymerase III termination sequences). Fusion of the DNA sequence to a tRNAimet deletion mutant such as Δ3′-5 would lead to the generation of a co-transcript from which subsequent processing of the tRNA leader at the 5′ portion of the fused transcript would be blocked. Control over processing may be of some biological use in engineered constructions, as suggested by properties of mRNA species bearing tRNA sequences as 5′ leaders in prokaryotes. Such “dual transcripts” code for several predominant bacterial proteins such as EF-Tu and may use the tRNA leaders as a means of stabilizing the transcript from degradation in vivo. The potential use of the tRNAimet gene as a “promoter leader” in eukaryotic systems has been realized recently in our laboratory. Fusion genes consisting of the deleted tRNAimet sequences contained on plasmids Δ 3′-4 and Δ 3′-5 in front of a promoter-less Herpes simplex type I thymidine kinase gene yield viral-specific enzyme resulting from RNA polymerase III dependent transcription in both X. Iaevis oocytes and somatic cells”. [References omitted].        
Sullenger et al., 1990 Cell 63, 601-619, describe over-expression of TAR-containing sequences using a chimeric tRNAimet-TAR transcription unit in a double copy (DC) murine retroviral vector.
Sullenger et al., 1990 Molecular and Cellular Bio. 10, 6512, describe expression of chimeric tRNA driven antisense transcripts. It indicates:                successful use of a tRNA-driven antisense RNA transcription system was dependent on the use of a particular type of retroviral vector, the double-copy (DC) vector, in which the chimeric tRNA gene was inserted in the viral LTR. The use of an RNA pol III-based transcription system to stably express high levels of foreign RNA sequences in cells may have other important applications. Foremost, it may significantly improve the ability to inhibit endogenous genes in eucaryotic cells for the study of gene expression and function, whether antisense RNA, ribozymes, or competitors of sequence-specific binding factors are used. tRNA-driven transcription systems may be particularly useful for introducing “mutations” into the germ line, i.e., for generating transgenic animals or transgenic plants. Since tRNA genes are ubiquitously expressed in all cell types, the chimeric tRNA genes may be properly expressed in all tissues of the animal, in contrast to the more idiosyncratic behavior of RNA pol II-based transcription units. However, homologous recombination represents a more elegant although, at present, very cumbersome approach for introducing mutations into the germ line. In either case, the ability to generate transgenic animals or plants carrying defined mutations will be an extremely valuable experimental tool for studying gene function in a developmental context and for generating animal models for human genetic disorders. In addition, tRNA-driven gene inhibition strategies may also be useful in creating pathogen-resistant livestock and plants. [References omitted.]        
Cotten and Birnstiel, 1989 EMBO Jrnl. 8, 3861, describe the use of tRNA genes to increase intracellular levels of ribozymes. The authors indicate that the ribozyme coding sequences were placed between the A and the B box internal promoter sequences of the Xenopus tRNAmet gene. They also indicate that the targeted hammerhead ribozymes were active in vivo.
Yu et al., 1993 Proc. Natl. Acad. Sci. USA 90, 5340, describe the use of a VAI promoter to express a hairpin ribozyme. The resulting transcript consisted of the first 104 nucleotides of the VAI RNA, followed by the ribozyme sequence and the terminator sequence. Lieber and Strauss, 1995 Mol. Cellular Bio. 15, 540, inserted a hammerhead ribozyme sequence in the central domain of a VAI RNA.